Analysis apparatus and method comprising auto-focusing means

ABSTRACT

The present invention relates to an analysis apparatus, in particular a spectroscopic analysis apparatus, for analyzing an object, such as blood of a patient, and a corresponding analysis method. To aim the confocal detection volume inside a blood vessel orthogonal polarized spectral imaging (OPS imaging) is used to locate blood capillaries in the skin. Image processing means (ipm) determining image characteristics, which indicate if an imaging system (img) for imaging the object is focused on the object (obj) to be analyzed, from a detected image. Said image processing means (ipm) are preferably adapted for determining the amplitudes of spatial frequencies corresponding to typical characteristics of the object (obj) from a detected image, or for determining the maximum contrast present in a detected image. Based on the determined image characteristics, auto-focusing means (afm) control the focusing means (mo) to change the focusing accordingly, whereafter the object is imaged and the same image characteristics are determined again from a the new image. This is preferably repeatedly done until the object (obj) substantially lies in a detection plane (dp) onto which the focusing means (mo) are focused. Thus, continuous autofocusing with high accuracy can be achieved.

The present invention relates to an analysis apparatus, in particular aspectroscopic analysis apparatus, for analyzing an object, such as theblood of a patient, and a corresponding analysis method. Further, theinvention relates to an optical focusing system for focusing on a targetpoint of an object.

In general, analysis apparatuses, such as spectroscopic analysisapparatuses, are used to investigate the composition of an object to beexamined, e.g. to measure the concentration of various analytes in bloodin vivo. In particular, analysis apparatuses employ an analysis, such asa spectroscopic decomposition, based on interaction of the matter of theobject with incident electromagnetic radiation, such as visible light,infrared or ultraviolet radiation.

A spectroscopic analysis apparatus comprising an excitation system and amonitoring system is known from WO 02/057759 A2 which is incorporatedherein by reference. The excitation system emits an excitation beam toexcite a target region during an excitation period. The monitoringsystem emits a monitoring beam to image the target region during amonitoring period. The excitation period and the monitoring periodsubstantially overlap. Hence the target region is imaged together withthe excitation, and an image is formed displaying both the target regionand the excitation area. On the basis of this image, the excitation beamcan be very accurately aimed at the target region.

The analysis method known from WO 02/057759 A2 for simultaneous imagingand spectral analysis of a local composition is done by separate lasersfor confocal video imaging and Raman excitation or by use of a singlelaser for combined imaging and Raman spectroscopic analysis. Orthogonalpolarized spectral imaging (OPS imaging), which is also described in WO02/057759 A2, is a simple, inexpensive and robust method to visualizeblood vessels close to the surface of organs which can also be used tovisualize blood capillaries in the human skin. Blood capillaries closeto the skin surface have a diameter of about 10 μm. Due to confocaldetection the source of collected Raman signals is well confined in allthree dimensions inside a spot of a size smaller than 2×2×8 μm³. Thisallows collecting Raman signals from blood without background signalfrom skin tissue if the focus is located in a blood capillary. This spotlocation is possible if the lateral position of the blood vessel as wellas the depth of the vessels below the skin surface are known with aresolution of 1 μm or better.

OPS imaging for blood vessel detection is also described in detail inEuropean patent application 03100689.3. The analysis apparatus describedtherein produces a contrast image in a contrast wavelength range and areference image in a reference wavelength range, said images beingcompared to accurately identify the target region, notably a capillaryblood vessel in the patient's skin.

Because of an effective back-illumination of blood vessels, OPS imagingis essentially a 2-dimensional technique. The only depth information isobtained by the influence of the amount of (de)focus on the images. Ifan objective with a numerical aperture (NA) higher than 0.8 is used, thedepth of field in skin is below 0.5 μm. Therefore, with accuratefocusing algorithms based on image analysis it is possible to find thedepth of the blood vessel.

Known auto-focusing methods are based on scanning the axial position ofthe objective focusing the imaging beam and the confocal excitation beamonto the object of interest, while measuring the value of a meritfunction to quantify the amount of (de)focus in the image. The bestfocus is found by optimizing the value of the merit function. In generalthere are many possibilities to change the focus position. For instance,one or two lenses in the objective can be moved (as in a photo camera)or the whole objective lens or another lens in the system can be moved.Also the shape of an optical element in the system can be changed, forexample an electro wetting optical element. However, if the object isnot known, the maximum of the merit function is also unknown. Therefore,the merit function provides only information about the amount of focusin relation to other focus positions.

Patients will move in lateral as well as in transversal directions.Therefore, continuous measurement and adjustment of the optimal locationof the confocal detection center is required. Transversal movements inthe image plane can easily be detected, whereas axial movements(perpendicular to the detection plane) are much more difficult todetect. A common method of detecting axial movement or defocus is bycontinuously moving the detection plane around the central best focusposition (so called wobbling). This can be done by moving the imagingobjective or another optical element in the imaging system. If the focusbecomes better in front or behind the central position, the centralposition of the objective is changed. In known systems the detectionvolume is located in the image plane. Therefore this detection volume isalso continuously moved around the best measurement position. This hasthe disadvantage that the confocal detection volume is located inside ablood vessel for only a fraction of time, and to avoid mixing of skinspectra with blood spectra, the intake of Raman signal has to be gated.This increases the time needed to collect sufficient Raman signal, whichis in case of continuous recording already at least 30 sec.

Further disadvantages are, that due to changes in the blood flow theshape and size of a capillary change continuously; so that comparingimages acquired at different times add uncertainty to the position ofbest focus. Additionally, the fact that more time is needed to collectsufficient Raman signal adds to the noise in the Raman spectrum becausemore dark current is acquired or because more readout noise is added.

It is therefore an object of the present invention to provide anoptimized analysis apparatus and a corresponding analysis method forimaging and spectroscopic analysis of an object which allow continuousaccurate auto-focusing of the excitation beam onto the object, inparticular a blood vessel, even during movements of the object withoutchanging the position of the detection volume continuously. Further, anoptical focusing system for focusing on a target point of an objectshall be provided, which system can, for instance, be applied in atracking system for continuously tracking the target point in a movingobject.

This object is achieved according to the present invention by ananalysis apparatus as claimed in claim 1 comprising:

an excitation system for emitting an excitation beam to excite a targetregion,

a monitoring system comprising a monitoring beam source for emitting amonitoring beam and an imaging system to image the target region,

a detection system for detecting scattered radiation from the targetregion generated by the excitation beam,

focusing means for focusing the excitation system, the monitoring systemand the detection system on a detection plane in the target region,

image processing means for determining image characteristics, whichindicate if the imaging system is focused on the object to be analyzed,from a detected image, and

auto-focusing means for controlling the focusing means to change thefocusing of the monitoring system, the excitation system and thedetection system based on the determined image characteristics, forcontrolling the monitoring system to image the target region and forcontrolling the image processing means to determine the imagecharacteristics from a detected image until the object substantiallylies in the detection plane.

The object is further solved by a corresponding analysis method asclaimed in claim 11. Preferred embodiments of the invention are definedin the dependent claims.

The invention is based on the idea to evaluate the detected image, todetermine certain image parameters and to conclude therefrom if theimaging system, and thus also the excitation system and the detectionsystem are focused on the object which shall be analyzed. The determinedimage characteristics are used to decide if the focusing needs to bechanged or not. If the object does not yet lie in the detection plane,or in other words, if the focusing is not yet sufficient, the focusingis changed whereafter a new image is detected and new imagecharacteristics are determined therefrom in order to again check if thefocusing is sufficient This recursive procedure can be executedcontinuously during analysis of the object in order to ensure that theRaman confocal detection volume can be continuously located exactlyinside the object of interest (such as a blood vessel).

Compared to other known auto-focusing techniques the present inventionprovides the advantage that the confocal detection volume iscontinuously located in the center of the object of interest, even ifthe object moves during the measurement. According to preferredembodiments no moving elements are required and a single microscopeobjective having a high numerical aperture can be used as focusingmeans. No continuous movement of the detection plane around the centralbest focus position (wobbling) is required. Another advantage of thepresent invention is that a simple, fast and robust (relative) focusmeasure is obtained which is required for many focusing methods, such asfor continuous tracking methods or for just finding the right depth of ablood vessel a single time (e.g. before a Raman measurement to locatethe depth of a capillary blood vessel).

Different image parameters are available which allow an indication ifthe imaging system is focused on the object of interest. According to apreferred embodiment as claimed in claim 2 the spatial frequenciescorresponding to typical characteristics of the object of interest, e.g.to typical diameters of blood vessels during in vivo analysis of blood,are determined from a detected image. Since in focus the amplitudes ofsuch spatial frequencies are maximally the focusing is changed until thedetermined amplitudes are maximally.

According to another preferred embodiment as claimed in claim 4 themaximum contrast present in a detected image and/or at one or more imageportions corresponding to the object or object portions, e.g. themaximum contrast present in a detected image between blood andsurrounding tissue, in particular at the edges of blood vessels, duringin vivo analysis of blood, are determined. Since in focus the contrastis maximally, the focusing is changed until the determined contrast ismaximally.

Preferred embodiments based on maximizing the contrast are defined independent claims 6 to 9.

It is preferred that the monitoring system is adapted for orthogonalpolarized spectral imaging as mentioned above and as described inW002/057759 A1 and in European patent application 03100689.3.

The invention can not only be used in an analysis apparatus as describedabove, but relates also to an optical focusing system for focusing on atarget point of an object, comprising a target system to be focused onthe target point, a monitoring system, focusing means, image processingmeans and auto-focusing means as claimed in claim 12. The invention canbe used in every system where an imaging system is used to locate andcontinuously track a target point, for example the focus of a laser beamor the detection volume of a spectroscopic system, continuously in 3dimensions at a specific position in a moving target. Examples include:(biomedical) laser surgery, laser cutting, laser welding, laser shaving,photodynamic therapy, remote sensing, and target and tracking inmilitary applications. Also the above described analysis apparatus couldbe regarded as including such an optical focusing system.

The invention will now be explained in more detail with reference toFIG. 1 which shows a graphic representation of an embodiment of ananalysis system according to the present invention.

FIG. 1 is a graphic representation of a preferred embodiment of ananalysis system in accordance with the invention. The analysis systemincludes an optical monitoring system (lso) for forming an optical imageof the object (obj) to be examined. In the present example the object(obj) is a piece of skin of the forearm of the patient to be examined.The analysis system also includes a multi-photon, non-linear, elastic orinelastic scattering optical detection system (ods) for spectroscopicanalysis of light generated in the object (obj) by a multi-photon,non-linear, elastic or inelastic optical process. The example shown inFIG. 1 utilizes in particular an inelastic Raman scattering detectionsystem (dsy) in the form of a Raman spectroscopy device. The termoptical encompasses not only visible light, but also ultravioletradiation and infrared, especially near-infrared radiation.

The monitoring system (lso) comprises a monitoring beam source (ls) foremitting a monitoring beam (irb) and an imaging system (img) for imagingthe target region, e.g. a blood vessel (V) in the upper dermis (D) ofthe patient's forearm (obj). The monitoring beam source (ls) in thisexample comprises a white light source (las), a lens (11) and aninterference filter (not shown) to produce light in the wavelengthregion of 560-570 nm. Further, a polarizer (p) for polarizing themonitoring beam (irb) is provided. The monitoring beam source (ls) isthus adapted for orthogonal polarized spectral imaging (OPS imaging).

In OPS imaging polarized light is projected by a microscope objective(mo) through a polarizing beam splitter (pbs) onto the skin (obj). Partof the light reflects directly from the surface (specular reflection).Another part penetrates into the skin where it scatters one or moretimes before it is absorbed or is re-emitted from the skin surface(diffuse reflection). In any of these scattering events the polarizationof the incident light is slightly changed. Light that is directlyreflected or penetrates only slightly into the skin will scatter onlyone or a few times before it is re-emitted, and will mostly retain itsinitial polarization. On the other hand, light that penetrates moredeeply into the skin undergoes multiple scattering events and iscompletely depolarized before re-emitted back towards the surface.

When looking at the object (obj) through a second polarizer or analyzer(a), oriented precisely orthogonal to that of the first polarizer (p),light reflected from the surface or the upper parts of the skin islargely suppressed, whereas light that has penetrated deep into the skinis mostly detected. As a result the image looks as if it wereback-illuminated. Because wavelengths below 590 nm are strongly absorbedby blood, the blood vessels appear dark in the OPS image.

Generally, an image is obtained using a monochrome CCD camera. Bloodvessels are separated from other absorbing structures be means of size,shape and movement of blood cells. The imaging system (img) used in thepresent embodiment comprises an analyzer (a) mentioned above forallowing only light having a polarization orthogonal to the light of thepolarized monitoring beam (irb) to pass which is reflected back throughthe polarizing beam splitter (pbs) from the object (obj). Said light isfurther focused by a lens (12) onto the CCD-camera (CCD).

The Raman spectroscopy device (ods) comprises an excitation system (exs)for emitting an excitation beam (exb) and a detection system (dsy) fordetection of Raman scattered signals from the target region. Theexcitation system (exs) can be constructed as a diode laser whichproduces the excitation beam in the form of an 785 nm infrared beam(exb). Of course other lasers can be used as excitation system as well.A system of mirrors and, for instance, an optical fiber conduct theexcitation beam (exb) to a dichroic mirror (f1) for conducting theexcitation beam (exb) along the monitoring beam (irb) to the microscopeobjective (mo) for focusing both beams onto the object (obj).

The dichroic mirror (f1) also separates the return (monitoring) beamfrom scattered Raman signals. While the reflected monitoring beam istransmitted to the imaging system (img), elastically and inelasticallyscattered Raman light from the object is reflected at the dichroicmirror (f1) and conducted back along the light path of the excitationbeam. Inelastically scattered Raman light is then reflected by anappropriate filter (f2) and directed along the Raman detection path inthe detection system (dsy) to the input of a spectrometer with a CCDdetector. The spectrometer with the CCD detector is incorporated intothe detector system (dsy) which records the Raman spectrum for awavelength range of 800 to 1050 nm. The output signal of thespectrometer with the CCD detector represents the Raman spectrum of theRaman scattered infrared light The signal output of the CCD detector isconnected to a spectrum display unit, for example a workstation thatdisplays the recorded Raman spectrum on a monitor. Also a calculationunit (e.g. a workstation) is provided to analyze the Raman spectrum andcalculate the concentration of one or more analytes.

Regarding further details of the analysis apparatus in general and thefunction thereof reference is made to the above mentioned WO 02/057759A1 and to European patent application 03100689.3.

To achieve continuous auto-focusing of the confocal Raman system (ods)in a blood vessel (V), image processing means (ipm) and auto-focusingmeans (afm) are provided. Such auto-focusing is required to locate ablood vessel and to aim the Raman system at this blood vessel. Sincepatients will move during a blood analysis in lateral (z) as well as intransversal (x, y) directions, continuous determination and adjustmentof the optimal location of the confocal detection center is required.Transversal movements can be easily detected by the imaging system,whereas axial movements are much more difficult to detect.

According to the present invention the image processing means (ipm)process an acquired image of the object (obj) and determine certainimage characteristics which indicate if the imaging system (img), andthus also the excitation system (exs) and the detection system (dsy) arefocused on the object (obj), in particular a blood vessel (V) or not,i.e. if the object of interest (the blood vessel) substantially lies inthe detection plane (dp) on which the microscope objective (mo) isfocused. Actually, only a relative focus measure can be determined ifthe object is not known, therefore, a focus measure is always comparedto a focus measure at a different position. The position with thehighest (or lowest) value of the focus measure is the position with bestfocus. Different image characteristics allow such an indication,preferably the amplitudes of spatial frequencies corresponding totypical diameters of blood vessels or the contsast between blood andsurrounding tissue. Based on the determined image characteristics theauto-focusing means (afm) control the microscope objective (mo) tochange the focusing thereof accordingly, i.e. to improve the focusing ifpossible. After this change the monitoring system (lso) is controlled totake another image of the object which is processed again by the imageprocessing means (ipm) in the same way in order to check if the focusinghas improved or not based on the same image characteristics, nowdetermined in the new image. This iterative procedure is executedcontinuously or at predetermined time intervals during the whole bloodanalysis to compensate for movements of the patient during the analysis.Another use is to determine the position of best focus before the Ramanmeasurement is started.

As mentioned above different image characteristics can be used. In thefollowing preferred image characteristics which are used for automaticfocusing in OPS imaging of blood vessels according to the presentinvention will be explained.

(1) A first preferred image characteristic is the typical dimensions ofblood vessels. It is known that the blood capillaries near the skinsurface have a typical diameter of 10 μm and a (visible) length of about50 μm. The 2-D spatial Fourier transform can be used to find the properfocus by maximizing the amplitudes of spatial frequencies withwavelengths equal or shorter than these typical dimensions. This can beused for monochromatic as well as bichromatic OPS imaging, bichromaticOPS imaging meaning OPS imaging with a contrast wavelength and areference wavelength as described in detail in European patentapplication 03100689.3.

(2) A second preferred image characteristic is the contrast betweenblood and surrounding tissue. Before discussing auto-focusing methodsbased on contrast, two general remarks have to be made. In bichromaticOPS imaging almost all structures visible after subtraction of the redand yellow/green image are blood vessels. Therefore any method based onmaximizing the contrast of an image automatically selects blood vessels.However, in monochromatic OPS imaging, blood vessels as well as otherstructures near the skin surface are visible. Therefore, care has to betaken to focus only on blood vessels. Other structures can be suppressedby averaging over a number of pixels and/or using a high-pass spatialFourier filter. In a monochromatic OPS imaging system the (preprocessed)image is used for image analysis and blood vessels appear as darkstructures on a light background. In bichromatic OPS imaging thedifference image (yellow/green minus red) is used for processing andblood vessels appear as light structures on a dark background.

Auto-focusing methods based on contrast can be divided into fourcategories. For all methods the average intensity of the image is keptconstant

-   (a) Maximize or minimize the intensity of a single pixel as a    function of depth. Light originating from outside the focal plane    (detection plane) will be spread over several pixels in the image.    By this spreading, the intensity of bright areas decreases, whereas    the intensity of dark regions increases. For monochromatic OPS    imaging, blood vessels are more dark compared to surrounding tissue,    hence blood vessels are in-focus if an image containing the darkest    pixel is obtained. For bichromatic OPS imaging, blood vessels are    more bright compared to surrounding tissue; hence blood vessels are    in-focus if an image containing the brightest pixel is obtained.    Mathematically this can be expressed as maximizing    (I_(i,j))_(max)(z) or minimizing (I_(i,j))_(min)(z). Here    (I_(i,j))_(max)(z) and (I_(i,j))_(min)(z) represent the intensity of    the pixel with maximum or minimum intensity when the imaging system    is focused at a depth z below the skin surface.-   (b) Maximize the spread in intensity of pixels as a function of    depth. Because light originating from outside the focal plane will    be spread over several pixels in the image, the spread in the    intensity distribution will decrease if blood vessels are out of    focus. The best focus can be obtained by maximizing    $\sum\limits_{i,j}{( {{I_{i,j}(z)} - {\overset{\_}{I}(z)}} )^{2}.}$    Here I_(i,j) (z) is the intensity measured at a pixel with    coordinates i,j and {overscore (I)}(z) is the intensity averaged    over all pixels when the imaging system is focused at a depth z    below the skin surface.-   (c) Maximize the average intensity difference between neighboring    pixels as a function of depth. Because light originating from    outside the focal plane will be spread over several pixels in the    image, the average intensity difference between adjacent pixels will    decrease if the vessels are outside the focal plane. The best focus    can be obtained by maximizing    ${\sum\limits_{i,j}( {{I_{i,j}(z)} - {I_{{i + 1},j}(z)}} )^{2}} + {( {{I_{i,j}(z)} - {I_{i,{j + 1}}(z)}} )^{2}.}$-   (d) Maximize absolute difference between neighboring pixels as a    function of depth. Instead of looking at the average intensity    difference between neighboring pixels in the whole image, the best    focus can be found by looking for the image with the largest    contrast between neighboring pixels. Mathematically this can be    expressed as maximizing    |I_(i,j)(z)−I_(i+1,j)(z)|+|I_(i,j)(z)−I_(i,j+1)(z)|.

Each of the methods described above has its own advantages anddisadvantages. Method (2 a) has the advantage that only a single pixel(belonging to a single blood vessel) is considered. By maximizing thecontrast, automatically the center of the thickest blood vessel close tothe skin surface is selected. This is also the position that can be usedmost conveniently for Raman spectroscopic analysis of blood. Method (2d) looks at the edge of a single blood vessel. Therefore at a singleblood vessel is focused, however, the optimum Raman measurement positionis located in the center of the vessel. Because only one or two pixelsare used in methods (2 a) and (2 d), these techniques are sensitive tonoise.

Methods (1), (2 b) and (2 c) use the whole image to find the best focus,and therefore are less sensitive to noise. However, they find the bestfocus of multiple blood vessels. If these vessels are not on the samedepth, the focal plane can lie in between the vessels. This problem canbe solved using a region of interest containing a single blood vesselfor focusing.

The contrast difference is only detectable for structures small comparedto the intensity spreading due to defocus. Therefore defocus can easilybe detected near the edge of structures. This is done in methods (2 c)and (2 d). The intensity in the center of the image of an object onlychanges due to defocus that causes a spread larger than the image of thestructure. Methods based on the maximum or minimum intensity (2 a) aretherefore less sensitive to defocus.

Another preferred embodiment will be a combination of auto-focusingalgorithms.

-   1. Use bichromatic OPS imaging to detect only structures that are    caused by absorption of blood-   2. Find the best average focus for multiple blood vessels by    maximizing the average intensity difference between neighboring    pixels (method 2 c).-   3. Select region of interest containing the image of a (part of a)    single blood vessel.-   4. Find the best focus for this vessel by-   a. maximizing the average intensity difference between neighboring    pixels (method 2 c) or-   b. minimize the intensity of a single pixel.

The choice for a or b depends on how the absolute intensity and theintensity difference depend on the amount of (de)focus.

The complete image can be used for auto-focusing. However, differentblood vessels or parts thereof lie at different depths below the skinsurface. Therefore, it is more accurate to use a region of interestaround the best Raman measurement position for auto-focusing. In adifferent application with higher quality images, an accuracy of 1% ofthe depth of focus can be achieved with the method according to thepresent invention. Thus, for automatic focusing of the Raman excitationbeam, the acquired accuracy in the order of 1 μm can be obtained.

In the above a monochromatic OPS imaging embodiment is described havinga white light source and a filter. Nevertheless, the invention can beapplied in many different embodiments including other monochromatic,bichromatic or multichromatic OPS imaging embodiments.

Compared to known auto-focusing techniques, the method according to thepresent invention has the advantage that the confocal detection volumecan be continuously located in the center of the blood vessel in whichthe blood shall be analyzed. The images that are evaluated arepreferably measured continuously. Further, the invention can be used asa simple, fast and robust (relative) method to obtain focus measurewhich can be used in many focusing methods, e.g. for just finding theright depth of a blood vessel a single time.

1. An analysis apparatus, in particular a spectroscopic analysisapparatus, for analyzing an object comprising: an excitation system foremitting an excitation beam to excite a target region, a monitoringsystem comprising a monitoring beam source for emitting a monitoringbeam and an imaging system to image the target region, a detectionsystem for detecting scattered radiation from the target regiongenerated by the excitation beam, focusing means for focusing theexcitation system, the monitoring system and the detection system on adetection plane in the target region, image processing means fordetermining image characteristics, which indicate if the imaging systemis focused on the object to be analyzed, from a detected image, andauto-focusing means for controlling the focusing means to change thefocusing of the monitoring system, the excitation system and thedetection system based on the determined image characteristics, forcontrolling the monitoring system to image the target region and forcontrolling the image processing means to determine the imagecharacteristics from a detected image until the object substantiallylies in the detection plane.
 2. An analysis apparatus as claimed inclaim 1, wherein said image processing means are adapted for determiningthe amplitudes of spatial frequencies corresponding to typicalcharacteristics of the object from a detected image and wherein saidauto-focusing means are adapted for controlling the focusing means tochange the focusing of the monitoring system, the excitation system andthe detection system based on the determined image characteristics, forcontrolling the monitoring system to repeatedly image the target regionand for controlling the image processing means to determine the imagecharacteristics from a detected image until the determined amplitudes ofspatial frequencies are maximally.
 3. An analysis apparatus as claimedin claim 2, wherein said analysis apparatus is adapted for in vivoanalysis of blood and wherein said image processing means are adaptedfor determining the amplitudes of spatial frequencies corresponding totypical diameters of blood vessels from a detected image.
 4. An analysisapparatus as claimed in claim 1, wherein said image processing means areadapted for determining the maximum contrast present in a detected imageand/or at one or more image portions corresponding to the object orobject portions and wherein said auto-focusing means are adapted forcontrolling the focusing means to change the focusing of the monitoringsystem, the excitation system and the detection system based on thedetermined image characteristics, for controlling the monitoring systemto repeatedly image the target region and for controlling the imageprocessing means to determine the image characteristics from a detectedimage until the determined contrast is maximally.
 5. An analysisapparatus as claimed in claim 4, wherein said analysis apparatus isadapted for in vivo analysis of blood and wherein said image processingmeans are adapted for determining the maximum contrast present in adetected image between blood and surrounding tissue, in particular atthe edges of blood vessels.
 6. An analysis apparatus as claimed in claim4, wherein said auto-focusing means are adapted for controlling thefocusing means to change the focusing of the monitoring system, theexcitation system and the detection system based on the determined imagecharacteristics, for controlling the monitoring system to repeatedlyimage the target region and for controlling the image processing meansto determine the image characteristics from a detected image until thedetermined the intensity of one or more pixels in the detected imageshow an extremum.
 7. An analysis apparatus as claimed in claim 4,wherein said auto-focusing means are adapted for controlling thefocusing means to change the focusing of the monitoring system, theexcitation system and the detection system based on the determined imagecharacteristics, for controlling the monitoring system to repeatedlyimage the target region and for controlling the image processing meansto determine the image characteristics from a detected image until thespread in intensity of pixels in the detected image is maximally.
 8. Ananalysis apparatus as claimed in claim 4, wherein said auto-focusingmeans are adapted for controlling the focusing means to change thefocusing of the monitoring system, the excitation system and thedetection system based on the determined image characteristics, forcontrolling the monitoring system to repeatedly image the target regionand for controlling the image processing means to determine the imagecharacteristics from a detected image until the average intensitydifference between neighboring pixels in the detected image ismaximally.
 9. An analysis apparatus as claimed in claim 4, wherein saidauto-focusing means are adapted for controlling the focusing means tochange the focusing of the monitoring system, the excitation system andthe detection system based on the determined image characteristics, forcontrolling the monitoring system to repeatedly image the target regionand for controlling the image processing means to determine the imagecharacteristics from a detected image until the absolute intensitydifference between neighboring pixels in the detected image ismaximally.
 10. An analysis apparatus as claimed in claim 1, wherein themonitoring system is adapted for orthogonal polarized spectral imaging,in particular for bichromatic orthogonal polarized spectral imaging. 11.An analysis method, in particular a spectroscopic analysis method, foranalyzing an object comprising the steps of: emitting an excitation beamby an excitation system to excite a target region, emitting a monitoringbeam by a monitoring system to image the target region by an imagingsystem, detecting scattered radiation from the target region generatedby the excitation beam by a detection system, focusing the excitationsystem, the monitoring system and the detection system on a detectionplane in the target region by a focusing means, determining imagecharacteristics, which indicate if the imaging system is focused on theobject to be analyzed, from a detected image, and controlling thefocusing means to change the focusing of the monitoring system, theexcitation system and the detection system based on the determined imagecharacteristics, controlling the monitoring system to image the targetregion, and controlling the image processing means to determine theimage characteristics from a detected image until the objectsubstantially lies in the detection plane.
 12. An optical focusingsystem for focusing on a target point of an object, comprising: a targetsystem to be focused on the target point, a monitoring system comprisinga monitoring beam source for emitting a monitoring beam and an imagingsystem to image the target region, focusing means for focusing thetarget system and the monitoring system on a detection plane in thetarget region, image processing means for determining imagecharacteristics, which indicate if the imaging system is focused on theobject to be analyzed, from a detected image, and auto-focusing meansfor controlling the focusing means to change the focusing of themonitoring system and the target system based on the determined imagecharacteristics, for controlling the monitoring system to image thetarget region and for controlling the image processing means todetermine the image characteristics from a detected image until theobject substantially lies in the detection planed.
 13. An opticaltracking system as claimed in claim 12, wherein said target systemcomprises a light beam generation means for emitting a light beam, inparticular a laser for emitting a laser beam, to be focused on thetarget point of the object.
 14. An optical tracking system as claimed inclaim 12, adapted for use in the field of laser surgery, laser cutting,laser welding, laser shaving, photodynamic therapy, radio therapy,remote sensing and target and tracking.